It is now widely anticipated that the present level of evaluation of mechanical function in biomaterials and tissue engineering studies is highly insufficient. For example of 205 analyzed articles on cartilage tissue engineering, mentioning of applied mechanical stimulation, only 29% has some quantified material properties [1]. Correct and detailed biomaterial testing is rather time-consuming and expertise to properly quantify non-elastic material behavior of tissue is also scarce in many dedicated biology labs [1]. The unfortunate consequence is that little is currently known about how specific culture regimes stimulate functional growth [2].
One of the essential information lacking is transient physical maturation of biomaterials and tissue engineering constructs. Mapping the material properties could guide the development of effective culture protocols, being particularly important in the design of biodegradable materials, where the rate of degradation should coincide with the rate of new matrix formation [1]. The quality of information expected by the user of such methods should be not only sufficiently rigorous to provide scientifically based evidence on the material or tissue, but also to provide acceptable correlations, trends and predictions which can be safely used in design, development and applications of biomaterials.
Conventional mechanical testing or characterization of the material itself, usually involves determination of strength, hardness, fatigue, coatings adhesion strength, etc. as well as so called materials properties like elastic (Young) modulus, shear modulus, viscosity, loss tangent (for dynamic loading), etc. Many biomaterials, including those for implants, are being nowadays tested under different mechanical loading schemes, specified by various standards. Besides conventional (tensile, bending etc.) tests for materials themselves, there are also dedicated tests for implant materials such as fatigue tests (e.g. ISO 14801 for dental implants). These tests are targeting on determination of a few parameters only, such as tensile strength, high-cycle fatigue limit, and they are mostly destructive. Their main purpose is to determine the practical limits of materials in service conditions from mechanical point of view only. Standard mechanical tests do not usually involve any kind of biological factors. Here and later, only tests which do not lead to clear destruction of the specimen, i.e. non-destructive evaluation, are being considered.
The data quality reported for the same material might be also confusing, as no exact information is given for conditioning changes, and usually no solid proof shown e.g. about suitability of the small strain theory or material linearity [1]. Such conditions are often assumed by default, despite it is of common knowledge that “elastic modulus” cannot be uniquely defined for material which does not follow linear elasticity model.
Biocompatibility and other biological type in vitro tests evaluate biomaterials' ability to work in vitro, such as ISO 10993. Tests are being carried out in respective culture wells or similar devices with only goal to access the effect of materials (in direct contact or via an extract) on living cells in static conditions. There are many attempts of simultaneous application of biological objects (cells cultures) and movable materials specimens [3].
These methods might be roughly classified into two categories: various bioreactors and combined testing devices.
The purpose of the first ones (bioreactor based methods) is mainly to provide conditions for tissue cell culturing (tissue engineering applications such as growing tissue grafts), and the mechanical forces there are poorly controlled—rotating flask, perfusion cells, etc. [3]. As a result, realistic stresses and strain acting on the material are not possible to evaluate, and usually reported “fluid shear stress” or other such properties are roughly assessed as for fluid flow in some equivalent size channel system [4]. Such an approach is limited by the laws of physics as these stress and strains cannot be measured in principle, but only calculated (only real forces and displacements can be measured directly). For example, in [5], a device design is described to provide micromotions on the material in the presence of osteoblast cells. However, this device is not able to mimic host conditions at the bone/implant interface, because it does not include proper microfluidics or microstrains and therefore does not produce experimental results which are relevant in the present invention.
Furthermore, the conditions for known biomaterial testing methods usually are not compatible with proper tissue engineering and cell cultivation applications. For example, applied mechanical stimulus is known to cause cellular toxicity, involving multiple yet unclear mechanisms in bone cells. Also increase of mechanical strain has been shown to increase cellular toxicity in osteoblasts [6]. Thus bioreactors are in principle incapable to measure and quantify properties of the biomaterial itself, whether or not it is combined with the cells or living tissues.
To match cell culturing conditions with assessment of biomaterials several kinds of composite devices and methods have been reported. With these devices it is attempted to improve the situation by placing the material specimens into more biologically controlled environment (a closed chamber or the like) with simultaneous application of mechanical stimulus via feeding probes or grips. This is usually implemented within a mechanical testing machine, requiring a substantial modification to ensure that correct stimulus is indeed transferred to the material.
In one example [7] several cell-laden specimens are being loaded into a sample holder and dynamically compressed at prescribed pattern aiming on simultaneous measurement of bioactivity of the cells and elastic modulus of the specimens. As several specimens are loaded at once, no individual strain and stress control is possible, and the determination of the properties of the materials is based on fitting deformation curves to an arbitrary theory (such as biphasic model). As reported by the authors themselves [7], this theory has failed to describe behavior of these materials. In summary, this method and the apparatus require many assumptions, new theories or independent experiments to extract true material data.
In another example [1], the deformation stimulus was applied for every sample individually in a test battery, but the resulting force was not possible to measure, and hence stress values were only approximated after the experiments. Furthermore, application of small elastic strain model to materials which clearly do not follow this rule has lead to simplifications which decrease the quality and question the relevance of the data.
In yet another example [8], even more specimens were simultaneously subjected to prescribed loading, however, making it impossible to directly evaluate measurable data, as the signals required attachment of magnets and resistance sensors, requiring every time specific non-linear calibration to convert voltage drop into force.
Similar version of this method [9] was again directed on increase of number of specimens but failed to get strain resolution, control of strain differences between the specimens, and faced substantial non-uniformity of the deformation (as it was dictated by the tallest specimen). Final data analysis was based on assumption of the statistical “strength of control” which was reported to influence results criteria significantly.
In another example (US Patent App. 2005/153436A1), a servocontrolled bioreactor system is shown, designed especially for orthopaedic tissue engineering products, and the main purpose of which is to grow cells constructs. The description of this patent application also underlines that stiffness of the ‘bioprosthesis’ being tested should match the native tissue for all operating ranges or frequencies, which is impossible to have in practice.
In yet another example (US Patent App. 2014/0295538A1), a device and a system for mechanical measurement of biomaterial are disclosed. That device and the method is applicable only to transparent soft materials, as they employ digital image correlation in the volume of the specimen via microscopy techniques, based on displacement of embedded nanoparticle markers. Whereas the method may give exact specific displacements of the markers, it fails to determine mechanical properties as its application requires biomaterial elastic modulus to be known.
There are also other examples (US Patent App. 2012/035742A1, 2011/136225A1, U.S. Pat. No. 6,107,081), which outline such combined methods and devices having the cell and tissue culturing as the main objective. Such methods are unable to evaluate most of the properties of biomaterials (for instance, when a specimen does not have cells seeded, the application of these methods becomes obsolete). With or without the cell cultures, time-invariant properties of the biomaterials cannot be assessed in these systems.
All these methods have intrinsic flaws in measurement precision as none of them is able to subtract the supporting platen stiffness contribution, empty sample holder correction, temperature variations, or effect of the intermediate layers introduced (resistance sensors, adhesives, magnets, etc.). Increasing number of specimens must be paid off with less read-outs—for example, in method described in [9] only approximate elastic modulus was possible to measure.
A special group of methods aims on high-throughput screening of various biomaterials, where the application of mechanical stimulus is foreseen and some response of the material to that stimulus is being measured. For example, U.S. Pat. No. 9,043,156 discloses a method for monitored application of mechanical force to samples using acoustic energy and mechanical parameter values extraction using mechanical response models. This method is based on ultrasonic excitation, i.e. sonic wave propagation through an unknown material and measurement of that wave (signal) attenuation in time. Despite being a non-destructive evaluation method, it however fails to identify realistic properties of a material, as it requires many assumptions (speed of sound in the material, non-linearity of the attenuation coefficient, unknown correction factors, needed uniformity of the specimen and constant density, etc.). This method also does not utilize the wave propagation theory and therefore requires from the user a pre-selection of some mechanical model of the material before making any calculations. In this method, only elastic modulus, relaxation time and viscosity coefficient can be assessed in as much as they are linked to some pre-determined materials models. Change of the model would lead to different set of these values. This leads to large errors (about 50%) and necessity of additional experiments to determine initially guessed parameters is required for such calculations. Furthermore, this method does not teach how to process the data if many parameters are unknown and the material does not follow linear model or is clearly inhomogeneous. Moreover, use of ultrasound, even for short duration, causes some dissipation of the pulse power into heat, locally affecting the material being tested.
In another example US 2011/013758 similar method is disclosed, related to measurement of rheological properties of a material or a biomaterial. This method also uses ultrasound excitation, having the same drawbacks as the method of U.S. Pat. No. 9,043,156, but it aims on minimizing the need for parameters by using ultrasound excitation in two dimensions. With use of harmonic signal excitation and using complex Fourier transform, this method is limited to components of viscoelastic properties of flowing media only and at very high frequencies (close to sound speed range). Ultrasound test for such materials reveals different materials properties than low-frequency measurements which is more relevant for biomaterials in implants. The values obtained with any ultrasound-based method are not time-invariant ones, they do not incorporate history of loading (being useful for fluid materials) and they cannot characterize the material to the extent foreseen in the present invention.
Another method of high-throughput screening of a material with application of mechanical stimulus is disclosed in the US Patent Application US2009/0088342. This method simultaneously applies a force or displacement to an array of specimens located at flexible (polymer) membrane via adjustable pressure of a fluid on the other side of that force-transfer membrane. The method however, is only feasible for very small samples, such as for microsystems, requiring use of flexible and optically transparent substrates, bonding by an adhesive (which properties and contribution to the signal are not known), and uses a very simplified elastic theory for membranes deflection. This requires additional numerical modeling because the strain and stress distributions cannot be measured without assumption of linear elasticity of the material and the substrate at the same time. The reported observed errors in strain of 50-90% vs. average do not allow a unique determination of any relevant material property, as every specimen is subjected to a non-uniform stimulus and with unknown contribution from the device and substrate themselves.
In another example of U.S. Pat. No. 9,683,267, a method of in vitro testing of a specimen is disclosed, aimed on creation of a proper mechano-regulative index in the specimen by means of introduction of a controlled size orifice in the specimen test chamber to have respectively controlled fluid flow in and out of the chamber, where the fluid is further being analysed. However, this method requires prior knowledge of the specimen's mechanical properties such as elastic modulus, requires that the specimen deformation is purely elastic and assumes full linearity of the material behavior as otherwise the mechano-regulative index is not possible to calculate. Thus this method is incapable for measurement of specimen properties.
Yet another example in U.S. Pat. No. 6,772,642 presents a high-throughput mechanical testing device, used for combinatory screening purposes of two or more specimens at once. Its application, however, is limited to flexible polymer substrates and a very simple linear elastic theory for membranes deformation. This method is silent about the outputs in case the specimen is not homogeneous, if it behaves in a non-linear way and/or undergoes some transformations affected by the loading history.
None of known or above presented mechanical, biomechanical or combined methods is capable to measure and evaluate time-invariant properties of biomaterials (whether with cells as ATMP or as a part of hybrid products) simultaneously in one test from a single specimen. There is no single mechanical test which is able to get simultaneously a spectrum of time-invariant materials functions including e.g. aggregate modulus, slope modulus, dynamic modulus, alpha-value spectrum, viscostiffness spectrum, permeability, permittivity, characteristic times, intrinsic modulus and viscosity spectra, effective channel size for fluid transport, etc., without application of the fluid pressure gradient and without assumption of some simplified material model.